EnteroBase in 2025: exploring the genomic epidemiology of bacterial pathogens.

This paper presents an update on the content, accessibility and analytical tools of the EnteroBase platform for web-based pathogen genome analysis. EnteroBase provides manually curated databases of genome sequence data and associated metadata from currently >1.1 million bacterial isolates, more recently including Streptococcus spp. and Mycobacterium tuberculosis, in addition to Salmonella,Escherichia/Shigella,Clostridioides,Vibrio,Helicobacter,YersiniaandMoraxella. We have implemented the genome-based detection of antimicrobial resistance determinants and the new bubble plot graphical tool for visualizing bacterial genomic population structures, based on pre-computed hierarchical clusters. Access to data and analysis tools is provided through an enhanced graphical user interface and a new application programming interface (RESTful API). EnteroBase is now being developed and operated by an international consortium, to accelerate the development of the platform and ensure the longevity of the resources built. EnteroBase can be accessed at https://enterobase.warwick.ac.uk as well as https://enterobase.dsmz.de.

Whispers in the wind: Face mask sampling for Mycobacterium tuberculosis detection in children with pulmonary tuberculosis.

BACKGROUND: Recently, face mask sampling (FMS) confirmed detection of Mycobacterium tuberculosis DNA from exhaled breath in adults with TB. To date, no study has evaluated the use of FMS to detect pulmonary Tuberculosis (TB) in children. We developed a method for FMS of M. tuberculosis-specific DNA in children and performed a clinical exploration to assess feasibility in children. METHODS: Face masks were spiked, analysed on GeneXpert-Ultra, qPCR, and tNGS. Children with pulmonary TB were asked to wear three modified FFP2 masks for 30 minutes as part of an exploratory clinical study. RESULTS: Experiments with H37Ra M. tuberculosis strain showed a limit of 95% detection of 3.75 CFU (4.85-3.11; 95%CI) on GeneXpert-Ultra. Ten children with pulmonary TB participated in the clinical study. M. tuberculosis-specific DNA was detected on none of the face masks. CONCLUSIONS: Paediatric FMS has a low limit of detection for M. tuberculosis-specific DNA in vitro. However, M. tuberculosis DNA was not detected in any of thirty masks worn by children with pulmonary TB. This suggests that FMS in this form may not be more effective for detecting M. tuberculosis in children with TB than existing methods.

Bedaquiline Resistance after Effective Treatment of Multidrug-Resistant Tuberculosis, Namibia.

Bedaquiline is currently a key drug for treating multidrug-resistant or rifampin-resistant tuberculosis. We report and discuss the unusual development of resistance to bedaquiline in a teenager in Namibia, despite an optimal background regimen and adherence. The report highlights the risk for bedaquiline resistance development and the need for rapid drug-resistance testing.

Challenging the gold standard: the limitations of molecular assays for detection of Mycobacterium tuberculosis heteroresistance.

OBJECTIVES: Heteroresistant infections are defined as infections in which a mixture of drug-resistant and drug-susceptible populations are present. In Mycobacterium tuberculosis (M. tb), heteroresistance poses a challenge in diagnosis and has been linked with poor treatment outcomes. We compared the analytical sensitivity of molecular methods, such as GeneXpert and whole genome sequencing (WGS) in detecting heteroresistance when compared with the 'gold standard' phenotypic assay: the agar proportion method (APM). METHODS: Using two rounds of proficiency surveys with defined monoresistant BCG strains and mixtures of susceptible/resistant M. tb, we determined the limit of detection (LOD) of known resistance associated mutations. RESULTS: The LOD for rifampin-R (RIF-R) detection was 1% using APM, 60% using GeneXpert MTB/RIF, 10% using GeneXpert MTB/RIF Ultra and 10% using WGS. While WGS could detect mutations beyond those associated with RIF resistance, the LOD for these other mutations was also 10%. Additionally, we observed instances where laboratories did not report resistance in the majority population, yet the mutations were present in the raw sequence data. CONCLUSION: The gold standard APM detects minority resistant populations at a lower proportion than molecular tests. Mycobacterium bovis BCG strains with defined resistance and extracted DNA from M. tb provided concordant results and can serve in quality control of laboratories offering molecular testing for resistance. Further research is required to determine whether the higher LOD of molecular tests is associated with negative treatment outcomes.

First detection of a Mycobacterium tuberculosis XDR clinical isolate harbouring an RpoB I491F mutation in a Ukrainian patient treated in Germany, October 2023.

This report documents the case of a Ukrainian patient infected with an extensively drug-resistant (XDR) lineage 2 Mycobacterium tuberculosis strain harbouring the rifampicin resistance mutation RpoB I491F. This mutation is not detected by routine molecular WHO-recommended rapid diagnostics, complicating the detection and treatment of these strains. The occurrence of such mutations underscores the need for enhanced diagnostic techniques and tailored treatment regimens, especially in eastern Europe where lineage 2 strains and XDR-tuberculosis are prevalent.

Limited Nosocomial Transmission of Drug-Resistant Tuberculosis, Moldova.

Applying whole-genome-sequencing, we aimed to detect transmission events of multidrug-resistant/rifampin-resistant strains of Mycobacterium tuberculosis complex at a tuberculosis hospital in Chisinau, Moldova. We recorded ward, room, and bed information for each patient and monitored in-hospital transfers over 1 year. Detailed molecular and patient surveillance revealed only 2 nosocomial transmission events.

Characterization of Two Novel Inhibitors of the Mycobacterium tuberculosis Cytochrome bc1 Complex.

Drug-resistant tuberculosis is a global health care threat calling for novel effective treatment options. Here, we report on two novel cytochrome bc1 inhibitors (MJ-22 and B6) targeting the Mycobacterium tuberculosis respiratory chain with excellent intracellular activities in human macrophages. Both hit compounds revealed very low mutation frequencies and distinct cross-resistance patterns with other advanced cytochrome bc1 inhibitors.

Design of Multidrug-Resistant Tuberculosis Treatment Regimens Based on DNA Sequencing.

BACKGROUND: Comprehensive and reliable drug susceptibility testing (DST) is urgently needed to provide adequate treatment regimens for patients with multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB). We determined whether next-generation sequencing (NGS) analysis of Mycobacterium tuberculosis complex isolates and genes implicated in drug resistance can guide the design of effective MDR/RR-TB treatment regimens. METHODS: NGS-based genomic DST predictions of M. tuberculosis complex isolates from MDR/RR-TB patients admitted to a TB reference center in Germany between 1 January 2015 and 30 April 2019 were compared with phenotypic DST results of mycobacteria growth indicator tubes (MGIT). Standardized treatment algorithms were applied to design individualized therapies based on either genomic or phenotypic DST results, and discrepancies were further evaluated by determination of minimal inhibitory drug concentrations (MICs) using Sensititre MYCOTBI and UKMYC microtiter plates. RESULTS: In 70 patients with MDR/RR-TB, agreement among 1048 pairwise comparisons of genomic and phenotypic DST was 86.3%; 76 (7.2%) results were discordant, and 68 (6.5%) could not be evaluated due to the presence of polymorphisms with yet unknown implications for drug resistance. Importantly, 549 of 561 (97.9%) predictions of drug susceptibility were phenotypically confirmed in MGIT, and 27 of 64 (42.2%) false-positive results were linked to previously described mutations mediating a low or moderate MIC increase. Virtually all drugs (99.0%) used in combination therapies that were inferred from genomic DST were confirmed to be susceptible by phenotypic DST. CONCLUSIONS: NGS-based genomic DST can reliably guide the design of effective MDR/RR-TB treatment regimens.

Microevolution of Mycobacterium tuberculosis Subpopulations and Heteroresistance in a Patient Receiving 27 Years of Tuberculosis Treatment in Germany.

Preexisting and newly emerging resistant pathogen subpopulations (heteroresistance) are potential risk factors for treatment failure of multi/extensively drug resistant (MDR/XDR) tuberculosis (TB). Intrapatient evolutionary dynamics of Mycobacterium tuberculosis complex (Mtbc) strains and their implications on treatment outcomes are still not completely understood. To elucidate how Mtbc strains escape therapy, we analyzed 13 serial isolates from a German patient by whole-genome sequencing. Sequencing data were compared with phenotypic drug susceptibility profiles and the patient's collective 27-year treatment history to further elucidate factors fostering intrapatient resistance evolution. The patient endured five distinct TB episodes, ending in resistance to 16 drugs and a nearly untreatable XDR-TB infection. The first isolate obtained, during the patient's 5th TB episode, presented fixed resistance mutations to 7 anti-TB drugs, including isoniazid, rifampin, streptomycin, pyrazinamide, prothionamide, para-aminosalicylic acid, and cycloserine-terizidone. Over the next 13 years, a dynamic evolution with coexisting, heterogeneous subpopulations was observed in 6 out of 13 sequential bacterial isolates. The emergence of drug-resistant subpopulations coincided with frequent changes in treatment regimens, which often included two or fewer active compounds. This evolutionary arms race between competing subpopulations ultimately resulted in the fixation of a single XDR variant. Our data demonstrate the complex intrapatient microevolution of Mtbc subpopulations during failing MDR/XDR-TB treatment. Designing effective treatment regimens based on rapid detection of (hetero) resistance is key to avoid resistance development and treatment failure.

Transmission patterns of rifampicin resistant Mycobacterium tuberculosis complex strains in Cameroon: a genomic epidemiological study.

BACKGROUND: Determining factors affecting the transmission of rifampicin (RR) and multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains under standardized tuberculosis (TB) treatment is key to control TB and prevent the evolution of drug resistance. METHODS: We combined bacterial whole genome sequencing (WGS) and epidemiological investigations for 37% (n = 195) of all RR/MDR-TB patients in Cameroon (2012-2015) to identify factors associated with recent transmission. RESULTS: Patients infected with a strain resistant to high-dose isoniazid, and ethambutol had 7.4 (95% CI 2.6-21.4), and 2.4 (95% CI 1.2-4.8) times increased odds of being in a WGS-cluster, a surrogate for recent transmission. Furthermore, age between 30 and 50 was positively correlated with recent transmission (adjusted OR 3.8, 95% CI 1.3-11.4). We found high drug-resistance proportions against three drugs used in the short standardized MDR-TB regimen in Cameroon, i.e. high-dose isoniazid (77.4%), ethambutol (56.9%), and pyrazinamide (43.1%). Virtually all strains were susceptible to fluoroquinolones, kanamycin, and clofazimine, and treatment outcomes were mostly favourable (87.5%). CONCLUSION: Pre-existing resistance to high-dose isoniazid, and ethambutol is associated with recent transmission of RR/MDR strains in our study. A possible contributing factor for this observation is the absence of universal drug susceptibility testing in Cameroon, likely resulting in prolonged exposure of new RR/MDR-TB patients to sub-optimal or failing first-line drug regimens.

Use of Whole Genome Sequencing for Mycobacterium tuberculosis Complex Antimicrobial Susceptibility Testing: From Sequence Data to Resistance Profiles.

Whole genome sequencing of Mycobacterium tuberculosis complex (MTBC) isolates has been shown to provide accurate predictions for resistance and susceptibility for many first- and second-line anti-tuberculosis drugs. However, bioinformatic pipelines and mutation catalogs to predict antimicrobial resistances in MTBC isolates are often customized and detailed protocols are difficult to access. Here, we provide a step-by-step workflow for the processing and interpretation of short-read sequencing data and give an overview of available analysis pipelines.

Use of Whole Genome Sequencing for Mycobacterium tuberculosis Complex Antimicrobial Susceptibility Testing.

Whole genome sequencing (WGS) is becoming an important diagnostic tool for antimicrobial susceptibility testing of Mycobacterium tuberculosis complex (MTBC) isolates in many countries. WGS protocols usually start with the preparation of a DNA-library: the critical first step in the process. A DNA-library represents the genomic content of a DNA sample and consists of unique short DNA fragments. Although available DNA-library protocols come with manufacturer instructions, details of the entire process, including quality controls, instrument parameters, and run evaluations, often need to be developed and customized by each laboratory to implement WGS technology effectively. Here, we provide a detailed workflow for a DNA-library preparation based on an adapted Illumina protocol optimized for the reduction of reagent costs.

Risk of tuberculosis transmission by children in Hamburg, Germany.

BACKGROUND: Data from a prospective molecular-epidemiologic study (1997-2021) in Hamburg, Germany, were evaluated to assess the transmission risk of Mycobacterium tuberculosis complex (Mtbc) by children <15 years in a low-incidence setting. METHODS: Isolates of Mtbc were genotyped whole genome sequencing, applying a core genome multilocus sequence typing scheme. Close contacts of culture-confirmed children were examined for latent Mtbc infections (LTBI) with particular focus on IGRA testing. RESULTS: Out of 3154 culture-confirmed tuberculosis (TB) cases, 79 (2.5%) were children <15 years. Of those, 52 (58%) had pulmonary TB. Genotyping revealed that 35 of the 52 children (67%) were epidemiologically confirmed secondary cluster members; all of their source cases were adults. Six immigrant children presented without a presumed source case; their TB diagnoses came on average 48 weeks (interquartile range [IQR] 71) after their arrival in Germany. Three German-born children were determined to have been infected by adult relatives while visiting their parents' home country. Of the 317 children's close contacts tested with QuantiFERON-TB Gold-In Tube for LTBI, only 21 (6.6%) were positive. Absent a history of prior exposure or immigration from a high-incidence country, none of the contacts of younger (<10 years) TB-afflicted children was latently infected, whereas 2 older children infected 12 of their contacts, children and adults. During a mean observational period of 551 weeks (IQR 735) on average, no secondary TB cases appeared. CONCLUSIONS: Children with pulmonary TB disease, especially those aged below 10 years, rarely transmit Mtbc to their close contacts in a low-incidence setting.

Transmission Dynamics of a Mycobacterium tuberculosis Complex Outbreak in an Indigenous Population in the Colombian Amazon Region.

Whole genome sequencing (WGS) has become the main tool for studying the transmission of Mycobacterium tuberculosis complex (MTBC) strains; however, the clonal expansion of one strain often limits its application in local MTBC outbreaks. The use of an alternative reference genome and the inclusion of repetitive regions in the analysis could potentially increase the resolution, but the added value has not yet been defined. Here, we leveraged short and long WGS read data of a previously reported MTBC outbreak in the Colombian Amazon Region to analyze possible transmission chains among 74 patients in the indigenous setting of Puerto Nariño (March to October 2016). In total, 90.5% (67/74) of the patients were infected with one distinct MTBC strain belonging to lineage 4.3.3. Employing a reference genome from an outbreak strain and highly confident single nucleotide polymorphisms (SNPs) in repetitive genomic regions, e.g., the proline-glutamic acid/proline-proline-glutamic-acid (PE/PPE) gene family, increased the phylogenetic resolution compared to a classical H37Rv reference mapping approach. Specifically, the number of differentiating SNPs increased from 890 to 1,094, which resulted in a more granular transmission network as judged by an increasing number of individual nodes in a maximum parsimony tree, i.e., 5 versus 9 nodes. We also found in 29.9% (20/67) of the outbreak isolates, heterogenous alleles at phylogenetically informative sites, suggesting that these patients are infected with more than one clone. In conclusion, customized SNP calling thresholds and employment of a local reference genome for a mapping approach can improve the phylogenetic resolution in highly clonal MTBC populations and help elucidate within-host MTBC diversity. IMPORTANCE The Colombian Amazon around Puerto Nariño has a high tuberculosis burden with a prevalence of 1,267/100,000 people in 2016. Recently, an outbreak of Mycobacterium tuberculosis complex (MTBC) bacteria among the indigenous populations was identified with classical MTBC genotyping methods. Here, we employed a whole-genome sequencing-based outbreak investigation in order to improve the phylogenetic resolution and gain new insights into the transmission dynamics in this remote Colombian Amazon Region. The inclusion of well-supported single nucleotide polymorphisms in repetitive regions and a de novo-assembled local reference genome provided a more granular picture of the circulating outbreak strain and revealed new transmission chains. Multiple patients from different settlements were possibly infected with at least two different clones in this high-incidence setting. Thus, our results have the potential to improve molecular surveillance studies in other high-burden settings, especially regions with few clonal multidrug-resistant (MDR) MTBC lineages/clades.

Implementation of targeted next-generation sequencing for the diagnosis of drug-resistant tuberculosis in low-resource settings: a programmatic model, challenges, and initial outcomes.

Targeted next-generation sequencing (tNGS) from clinical specimens has the potential to become a comprehensive tool for routine drug-resistance (DR) prediction of Mycobacterium tuberculosis complex strains (MTBC), the causative agent of tuberculosis (TB). However, TB mainly affects low- and middle-income countries, in which the implementation of new technologies have specific needs and challenges. We propose a model for programmatic implementation of tNGS in settings with no or low previous sequencing capacity/experience. We highlight the major challenges and considerations for a successful implementation. This model has been applied to build NGS capacity in Namibia, an upper middle-income country located in Southern Africa and suffering from a high-burden of TB and TB-HIV, and we describe herein the outcomes of this process.

High fluoroquinolone resistance proportions among multidrug-resistant tuberculosis driven by dominant L2 Mycobacterium tuberculosis clones in the Mumbai Metropolitan Region.

BACKGROUND: Multidrug-resistant (MDR) Mycobacterium tuberculosis complex (MTBC) strains are a serious health problem in India, also contributing to one-fourth of the global MDR tuberculosis (TB) burden. About 36% of the MDR MTBC strains are reported fluoroquinolone (FQ) resistant leading to high pre-extensively drug-resistant (pre-XDR) and XDR-TB (further resistance against bedaquiline and/or linezolid) rates. Still, factors driving the MDR/pre-XDR epidemic in India are not well defined. METHODS: In a retrospective study, we analyzed 1852 consecutive MTBC strains obtained from patients from a tertiary care hospital laboratory in Mumbai by whole genome sequencing (WGS). Univariate and multivariate statistics was used to investigate factors associated with pre-XDR. Core genome multi locus sequence typing, time scaled haplotypic density (THD) method and homoplasy analysis were used to analyze epidemiological success, and positive selection in different strain groups, respectively. RESULTS: In total, 1016 MTBC strains were MDR, out of which 703 (69.2%) were pre-XDR and 45 (4.4%) were XDR. Cluster rates were high among MDR (57.8%) and pre-XDR/XDR (79%) strains with three dominant L2 (Beijing) strain clusters (Cl 1-3) representing half of the pre-XDR and 40% of the XDR-TB cases. L2 strains were associated with pre-XDR/XDR-TB (P < 0.001) and, particularly Cl 1-3 strains, had high first-line and FQ resistance rates (81.6-90.6%). Epidemic success analysis using THD showed that L2 strains outperformed L1, L3, and L4 strains in short- and long-term time scales. More importantly, L2 MDR and MDR + strains had higher THD success indices than their not-MDR counterparts. Overall, compensatory mutation rates were highest in L2 strains and positive selection was detected in genes of L2 strains associated with drug tolerance (prpB and ppsA) and virulence (Rv2828c). Compensatory mutations in L2 strains were associated with a threefold increase of THD indices, suggesting improved transmissibility. CONCLUSIONS: Our data indicate a drastic increase of FQ resistance, as well as emerging bedaquiline resistance which endangers the success of newly endorsed MDR-TB treatment regimens. Rapid changes in treatment and control strategies are required to contain transmission of highly successful pre-XDR L2 strains in the Mumbai Metropolitan region but presumably also India-wide.

Whole genome sequencing-based drug resistance predictions of multidrug-resistant Mycobacterium tuberculosis isolates from Tanzania.

Background: Rifampicin- or multidrug-resistant (RR/MDR) Mycobacterium tuberculosis complex (MTBC) strains account for considerable morbidity and mortality globally. WGS-based prediction of drug resistance may guide clinical decisions, especially for the design of RR/MDR-TB therapies. Methods: We compared WGS-based drug resistance-predictive mutations for 42 MTBC isolates from MDR-TB patients in Tanzania with the MICs of 14 antibiotics measured in the Sensititre™ MycoTB assay. An isolate was phenotypically categorized as resistant if it had an MIC above the epidemiological-cut-off (ECOFF) value, or as susceptible if it had an MIC below or equal to the ECOFF. Results: Overall, genotypically non-wild-type MTBC isolates with high-level resistance mutations (gNWT-R) correlated with isolates with MIC values above the ECOFF. For instance, the median MIC value (mg/L) for rifampicin-gNWT-R strains was >4.0 (IQR 4.0-4.0) compared with 0.5 (IQR 0.38-0.50) in genotypically wild-type (gWT-S, P < 0.001); isoniazid-gNWT-R >4.0 (IQR 2.0-4.0) compared with 0.25 (IQR 0.12-1.00) among gWT-S (P = 0.001); ethionamide-gNWT-R 15.0 (IQR 10.0-20.0) compared with 2.50 (IQR; 2.50-5.00) among gWT-S (P < 0.001). WGS correctly predicted resistance in 95% (36/38) and 100% (38/38) of the rifampicin-resistant isolates with ECOFFs >0.5 and >0.125 mg/L, respectively. No known resistance-conferring mutations were present in genes associated with resistance to fluoroquinolones, aminoglycosides, capreomycin, bedaquiline, delamanid, linezolid, clofazimine, cycloserine, or p-amino salicylic acid. Conclusions: WGS-based drug resistance prediction worked well to rule-in phenotypic drug resistance and the absence of second-line drug resistance-mediating mutations has the potential to guide the design of RR/MDR-TB regimens in the future.

Rapid molecular diagnostics of tuberculosis resistance by targeted stool sequencing.

BACKGROUND: Stool is an important diagnostic specimen for tuberculosis in populations who struggle to provide sputum, such as children or people living with HIV. However, the culture of Mycobacterium tuberculosis (M. tuberculosis) complex strains from stool perform poorly. This limits the opportunity for phenotypic drug resistance testing with this specimen. Therefore, reliable molecular methods are urgently needed for comprehensive drug resistance testing on stool specimens. METHODS: We evaluated the performance of targeted next-generation sequencing (tNGS, Deeplex® Myc-TB) for the detection of mutations associated with M. tuberculosis complex drug resistance on DNA isolated from stool specimens provided by participants from a prospective cohort of patients treated for tuberculosis in Eswatini (n = 66; 56 with and 10 participants without M. tuberculosis complex DNA detected in stool by real-time quantitative PCR), and an independent German validation cohort of participants with culture-confirmed tuberculosis (n = 21). RESULTS: The tNGS assay detected M. tuberculosis complex DNA in 38 of 56 (68%) samples; for 28 of 38 (74%) samples, a full M. tuberculosis complex drug resistance prediction report was obtained. There was a high degree of concordance with sputum phenotypic drug susceptibility results (κ = 0.82). The ability to predict resistance was concentration-dependent and successful in 7/10 (70%), 18/25 (72%), and 3/21 (14%) of samples with stool PCR concentration thresholds of > 100 femtogram per microliter (fg/µl), 1 to 100 fg/µl, and < 1 fg/µl, respectively (p = 0.0004). The German cohort confirmed these results and demonstrated a similarly high concordance between stool tNGS and sputum phenotypic drug susceptibility results (κ = 0.84). CONCLUSIONS: tNGS can identify drug resistance from stool provided by tuberculosis patients. This affords the opportunity to obtain critical diagnostic information for tuberculosis patients who struggle to provide respiratory specimens.

Origin and Global Expansion of Mycobacterium tuberculosis Complex Lineage 3.

Mycobacterium tuberculosis complex (MTBC) Lineage 3 (L3) strains are abundant in world regions with the highest tuberculosis burden. To investigate the population structure and the global diversity of this major lineage, we analyzed a dataset comprising 2682 L3 strains from 38 countries over 5 continents, by employing 24-loci mycobacterial interspersed repetitive unit-variable number of tandem repeats genotyping (MIRU-VNTR) and drug susceptibility testing. We further combined whole-genome sequencing (WGS) and phylogeographic analysis for 373 strains representing the global L3 genetic diversity. Ancestral state reconstruction confirmed that the origin of L3 strains is located in Southern Asia and further revealed multiple independent introduction events into North-East and East Africa. This study provides a systematic understanding of the global diversity of L3 strains and reports phylogenetic variations that could inform clinical trials which evaluate the effectivity of new drugs/regimens or vaccine candidates.

Implementation of whole genome sequencing for tuberculosis diagnostics in a low-middle income, high MDR-TB burden country.

Whole genome sequencing (WGS) is revolutionary for diagnostics of TB and its mutations associated with drug-resistances, but its uptake in low- and middle-income countries is hindered by concerns of implementation feasibility. Here, we provide a proof of concept for its successful implementation in such a setting. WGS was implemented in the Kyrgyz Republic. We estimated needs of up to 55 TB-WGS per week and chose the MiSeq platform (Illumina, USA) because of its capacity of up to 60 TB-WGS per week. The project's timeline was completed in 93-weeks. Costs of large equipment and accompanying costs were 222,065 USD and 8462 USD, respectively. The first 174 WGS costed 277 USD per sequence, but this was skewed by training inefficiencies. Based on real prices and presuming optimal utilization of WGS capacities, WGS costs could drop to 167 and 141 USD per WGS using MiSeq Reagent Kits v2 (500-cycles) and v3 (600-cycles), respectively. Five trainings were required to prepare the staff for autonomous WGS which cost 48,250 USD. External assessment confirmed excellent performance of WGS by the Kyrgyz laboratory in an interlaboratory comparison of 30 M. tuberculosis genomes showing complete agreeance of results.